(−)-³H-desmethoxyverapamil labelling of putative calcium channels in brain: autoradiographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazem binding sites

Summary

The optically pure phenylalkylamine Ca²⁺-antagonist (−)-³H-desmethoxyverapamil has been used to directly label putative Ca²⁺ channels in thaw-mounted guinea-pig brain sections and in hippocampus homogenates. The autoradiographic distribution of (−)-³H-desmethoxyverapamil binding shows the highest density of binding sites in the molecular layer of the hippocampus, medium levels in the cerebral cortex and low levels in the cerebellum. This anatomical distribution is the same as that found for 1,4-dihydropyridine binding sites, labelled by ³H-nimodipine. (−)-³H-desmethoxyverapamil binds to hippocampus membranes with a K _D value of 1.6±0.2 nmol/l and a B _max of 870±175 fmol per mg of protein. Binding is stereospecifically inhibited by the phenylalkylamines desmethoxyverapamil, D-600 and verapamil. 1,4-Dihydropyridines regulate (−)-³H-desmethoxyverapamil binding in a negative heterotropic allosteric manner, depending on the availability of free divalent cations. The potency series of phenylalkylamine Ca²⁺ antagonists in inhibiting high affinity (−)-³H-desmethoxyverapamil binding to hippocampus membranes and the allosteric regulation by chemically different classes of Ca²⁺ antagonists suggest that the (−)-³H-desmethoxyverapamil binding sites in hippocampus are associated with putative Ca²⁺ channels.